Blood coagulation is a process consisting of a complex interaction of various blood components (or factors) that eventually gives rise to a fibrin clot. Generally, the blood components, which participate in what has been referred to as the coagulation “cascade,” are enzymatically inactive proteins (proenzymes or zymogens) that are converted to proteolytic enzymes by the action of an activator (which itself is an activated clotting factor). Coagulation factors that have undergone such a conversion are generally referred to as “active factors,” and are designated by the addition of the letter “a” to the name of the coagulation factor (e.g., activated Factor VII is designated as Factor VIIa or FVIIa).
Normally, initiation of the haemostatic process is mediated by the formation of a complex between tissue factor and Factor VIIa. This complex then converts Factors IX (FIX) and X (FX) to their active forms. Factor Xa (FXa) converts limited amounts of prothrombin to thrombin on the tissue factor-bearing cell. Thrombin activates platelets and Factors V (FV) and VIII (FVIII) into Factors Va (FVa) and VIIIa (FVIIIa), both cofactors in the further process leading to the full thrombin burst. This process includes generation of Factor Xa by Factor IXa (FIXa) (in complex with Factor VIIIa) and occurs on the surface of activated platelets. Thrombin finally converts fibrinogen to fibrin resulting in formation of a fibrin clot. In recent years Factor VII and tissue factor have been found to be the main initiators of blood coagulation.
Factor VII is a plasma glycoprotein that circulates in blood as a single-chain zymogen. The zymogen has marginal catalytic activity. Single-chain Factor VII may be converted to two-chain Factor VIIa by Factor Xa, Factor XIIa, Factor IXa, Factor VIIa or thrombin in vitro. Factor Xa is believed to be the major physiological activator of Factor VII. Like several other plasma proteins involved in hemostasis, Factor VII is dependent on Vitamin K for its activity, which is required for the gamma-carboxylation of multiple glutamic acid residues that are clustered close to the amino terminus of the protein. These gamma-carboxylated glutamic acids are required for the metal ion-induced interaction of Factor VII with phospholipids. The conversion of zymogen Factor VII into the activated two-chain molecule occurs by cleavage of an internal Arg152-Ile153 peptide bond. Additionally, it is well known that high concentrations of Factor VII lead to autoactivation in vitro. In the presence of tissue factor, phospholipids and calcium ions, the two-chain Factor VIIa rapidly activates Factor X or Factor IX by limited proteolysis.
The gene coding for human FVII (hFVII) has been mapped to chromosome 13 at q34-qter 9 (de Grouchy et al., Hum Genet 1984; 66:230-233). It contains nine exons and spans 12.8 Kb (O'Hara et al., Proc Natl Acad Sci USA 1987; 84:5158-5162). The gene organization and protein structure of FVII are similar to those of other vitamin K-dependent procoagulant proteins, with exons 1a and 1b encoding for signal sequence; exon 2 the propeptide and GLA domain; exon 3 a short hydrophobic region; exons 4 and 5 the epidermal growth factor-like domains; and exon 6 through 8 the serine protease catalytic domain (Yoshitake et al., Biochemistry 1985; 24: 3736-3750).
Factor IX (Christmas factor) is the zymogen of a serine protease active in normal hemostasis and the enzymatic activity requires carboxylation of specific glutamic acid residues. Factors IX, X, VII and protein C are closely related paralogs of the same family of serine proteases, with a high degree of amino acid sequence identity and intron-exon arrangement of the genes coding for these proteins. These closely related proteins have a similar structure of functional domains from the amino to carboxyl terminus to include a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains, an activation peptide and the catalytic domain. Protein S is a 666 amino acid, vitamin K-dependent protein with a GLA domain, 4 EGF-like domains, a thrombin sensitive region and 2 laminin domains.
The vitamin K dependent coagulation plasma proteins contain a GLA domain that functions as the site of protein attachment to membranes and the GLA domain is highly conserved among the various coagulation proteins. Despite their similarity, the GLA domains exhibit a wide range of affinities for phospholipid, with the GLA domain of Protein S having the highest affinity for phospholipids. (Ellison et al., Biochemistry, 1998; 37:7997-8003), (McDonald et al., Biochemistry 1997; 36:5120-27).
It is often desirable to stimulate or improve the coagulation cascade in a subject. Factor VIIa has been used to control bleeding disorders that have several causes such as clotting factor deficiencies (e.g., hemophilia A and B or deficiency of coagulation Factors XI or VII) or clotting factor inhibitors. Factor VIIa has also been used to control excessive bleeding occurring in subjects with a normally functioning blood clotting cascade (no clotting factor deficiencies or inhibitors against any of the coagulation factors). Such bleeding may, for example, be caused by a defective platelet function, thrombocytopenia or von Willebrand's disease.
Bleeding is also a major problem in connection with surgery and other forms of trauma. For example, Factor VII has been used extensively for treating soldiers wounded in Iraq and Afghanistan. (Perkins J G, et al. The Journal of Trauma. 2007; 62: 1095-9; discussion 9-101). Its use has been credited with saving many lives, but as with most medical treatments, there are side effects, such as stroke or other thrombotic events after treatment. The overall impression of physicians using FVIIa, however, is that its use has saved many more lives than it has lost. Perhaps the best indication of this is that in previous wars approximately 30 percent of the wounded died of their injuries, while the number in the current Gulf war has been reduced to about 10 percent. (Gawande A, et al., N Engl J Med. 2004; 351: 2471-5).
Studies of transgenic hemophilia B mice expressing factor VIIa have demonstrated that continuous Factor VIIa expression at low levels (below 1.5 μg/ml) restores clotting activity in hemophilia B mice. Levels of factor VIIa in wild type or hemophilia B mice above 2 μg/ml, however, led to thromboses in the heart and lungs; both the heart and lungs are sites of high tissue factor expression. This suggests that the high levels of factor VIIa in the circulation induce thrombosis when they contact tissue factor exposed upon vessel injury in the heart and lungs. (Margaritas et al., J. Clin. Invest. 2004; 113:1025-31). Furthermore, studies have shown that vector mediated gene transfer of canine Factor VIIa in hemophilic dogs is both safe and effective in the short and medium term. (Margaritas et al., Gene Therapy 2009; 113:3682-3689).
Warnings relating to treatment with Factor VII are currently proposed for products seeking regulatory approval. For example, The European Medicines Agency, Human Medicines Evaluation Unit recommends that current Factor VII therapies carry a warning of the risk of thrombosis and disseminated intravascular coagulation, particularly in situations where the Factor VII is to be administered to patients with a history of coronary heart disease or liver disease, post-operative patients, neonates and those at risk from thrombosis and disseminated intravascular coagulation. See, e.g., Core SPC for Human Plasma Derived Coagulation Factor VII Products (CPMP/BPWG/2048/01), July, 2004.
Previously, it has been shown that the EGF-1 domain of Factor VIIa plays a critical role in the affinity of Factor VIIa for tissue factor. Using both a synthetic substrate and Factor X, in both the presence and absence of tissue factor, Factor VIIa polypeptides with a Factor IX EGF-1 domain had lower catalytic activity than the wild type Factor VIIa. (Jin et al., Biochemistry, 1999, 28:1185-92). At first glance, this would seem to obviate the use of chimeric constructs for treating bleeding; however, Monroe (British Journal of Haematology 1997; 99:542-549) has proposed that the mechanism of FVIIa in treating hemophilia and bleeding is tissue factor independent. Opinion in the art, however, is divided as to whether Factor VIIa is not active independent of tissue factor.
Commercial preparations of human recombinant FVIIa are sold as NovoSeven® and NovoSeven® RT. NovoSeven® and NovoSeven® RT are indicated for the treatment of bleeding episodes in hemophilia A or B patients and are the only rFVIIa for treatment of bleeding episodes available on the market. Recently, it has been demonstrated that NovoSeven® may bind to rehydrated lyophilized platelets, which could be administered in combination to localize the Factor VII to a site of injury. (Fischer et al., Platelets, 2008; 19:182-91). Additionally, it has been shown that selective PEGylation of Factor VII may increase plasma half-life, (Stennicke et al., Thromb. Haemost, 2008; 100:920-28), and that a recombinant human Factor VII, with 3 amino acid substitutions has an increased activity on the surface of platelets. (Moss et al., J. Thromb. Haemost., 2009; 7:299-305). PEGylation of the chimeric Factor VIIa molecules of the present invention are expected to work in a similar manner to increase plasma half-life. Likewise, other modifications of proteins known in the art, such as covalent attachment of non-polypeptide moieties to form conjugates, e.g., glycosylation, are expected to function in a similar manner in the chimeric Factor VIIa molecules of the present invention, i.e., the properties imparted to a protein by the covalent attachment of a non-polypeptide moiety are expected to be imparted to the chimeric Factor VII a molecules.
There is a need for variants of Factor VIIa having high coagulant activity that can be administered at relatively low doses, and variants which produce fewer undesirable side effects such as thrombotic complications, associated with available therapies.